Stéphane Grégoire

Short-Term Long Chain Omega3 Diet Protects from Neuroinflammatory Processes and Memory Impairment in Aged Mice

Regular consumption of food enriched in omega3 polyunsaturated fatty acids (ω3 PUFAs) has been shown to reduce risk of cognitive decline in elderly, and possibly development of Alzheimers disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most likely active components of ω3-rich PUFAs diets in the brain. We therefore hypothesized that exposing mice to a DHA and EPA enriched diet may reduce neuroinflammation and protect against memory impairment in aged mice. For this purpose, mice were exposed to a control diet throughout life and were further submitted to a diet enriched in EPA and DHA during 2 additional months. Cytokine expression together with a thorough analysis of astrocytes morphology assessed by a 3D reconstruction was measured in the hippocampus of young (3-month-old) and aged (22-month-old) mice. In addition, the effects of EPA and DHA on spatial memory and associated Fos activation in the hippocampus were assessed. We showed that a 2-month EPA/DHA treatment increased these long-chain ω3 PUFAs in the brain, prevented cytokines expression and astrocytes morphology changes in the hippocampus and restored spatial memory deficits and Fos-associated activation in the hippocampus of aged mice. Collectively, these data indicated that diet-induced accumulation of EPA and DHA in the brain protects against neuroinflammation and cognitive impairment linked to aging, further reinforcing the idea that increased EPA and DHA intake may provide protection to the brain of aged subjects.

Nutrition for the Eye: Different Susceptibility of the Retina and the Lacrimal Gland to Dietary Omega-6 and Omega-3 Polyunsaturated Fatty Acid Incorporation

The purpose of this study was to compare the susceptibility of the retina and the exorbital lacrimal gland to dietary supplies of long-chain omega-3 (ω3) and omega-6 (ω6) polyunsaturated fatty acids (LC-PUFAs). Male Wistar rats were fed a 5% lipid diet containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA or (4) a balanced diet deprived of EPA, DHA and GLA for 3 months. Lipids were extracted from plasma phospholipids, retina and exorbital lacrimal gland, and fatty acid composition was determined by gas chromatography. Dietary supplementation with EPA and DHA increased ω3 PUFA levels in plasma phospholipids as well as in the retina and the exorbital lacrimal gland. By contrast, GLA supplementation favored ω6 PUFA incorporation, and particularly the incorporation of the end-chain ω6 product, docosapentaenoic acid (DPA), into all tissues. Supplementation with EPA, DHA and GLA increased the levels of DHA, EPA and dihomo-GLA (dGLA), whereas arachidonic acid (AA) was unchanged and DPA decreased in the retina and the lacrimal gland. The ability of both tissues to incorporate PUFAs from blood was evaluated. The results showed that the retina was more selective than the lacrimal gland for EPA. In spite of the different susceptibility of the retina and the lacrimal gland to dietary PUFAs, these results suggest that the concomitant use of dietary ω3 and ω6 PUFAs may be useful in modulating inflammation in both tissues.

Decreasing dietary linoleic acid promotes long chain omega-3 fatty acid incorporation into rat retina and modifies gene expression.

Age-related macular degeneration (AMD) may be partially prevented by dietary habits privileging the consumption of ω3 long chain polyunsaturated fatty acids (ω3s) while lowering linoleic acid (LA) intake. The present study aimed to document whether following these epidemiological guidelines would enrich the neurosensory retina and RPE with ω3s and modulate gene expression in the neurosensory retina. Rat progenitors and pups were fed with diets containing low or high LA, and low or high ω3s. After scotopic single flash and 8-Hz-Flicker electroretinography, rat pups were euthanized at adulthood. The fatty acid profile of the neurosensory retina, RPE, liver, adipose tissue and plasma was analyzed using gas chromatography. Gene expression was analyzed with real-time PCR in the neurosensory retina. Diets rich in ω3s efficiently improved the incorporation of ω3s into the organs and tissues. This raising effect was magnified by lowering LA intake. Compared to a diet with high LA and low ω3s, low LA diets significantly upregulated LDL-receptor gene expression. Similar but not significant upregulation of CD36, ABCA1, ALOX5 and ALOX12 gene expression was observed in rats fed with low LA. No effect was observed on retinal function. Increasing the intake in ω3s and lowering LA improved the enrichment with ω3s of the tissues, including the neurosensory retina and RPE, and upregulated genes involved in lipid trafficking in the neurosensory retina. Those results consistently reinforced the beneficial role of ω3s in the prevention of AMD, especially when the diet contained low levels of LA, as suggested from epidemiological data.

Modulation of brain PUFA content in different experimental models of mice

The relative amounts of arachidonic acid (AA) and docosahexaenoic acid (DHA) govern the different functions of the brain. Their brain levels depend on structures considered, on fatty acid dietary supply and the age of animals. To have a better overview of the different models available in the literature we here compared the brain fatty acid composition in various mice models (C57BL/6J, CD1, Fat-1, SAMP8 mice) fed with different n-3 PUFA diets (deficient, balanced, enriched) in adults and aged animals. Our results demonstrated that brain AA and DHA content is 1) structure-dependent; 2) strain-specific; 3) differently affected by dietary approaches when compared to genetic model of PUFA modulation; 4) different in n-3 PUFA deficient aged C57BL6/J when compared to SAMP8 mouse model of aging. From these experiments, we highlight the difficulty to compare results obtained in different mouse models, different strains, different brain regions and different ages.

INHIBITION OF FATTY ACID DESATURASES IN Drosophila melanogaster LARVAE BLOCKS FEEDING AND DEVELOPMENTAL PROGRESSION

Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program.

Detection of lactobacillic acid in low erucic rapeseed oil - a note of caution when quantifying cyclic fatty acid monomers in vegetable oils.

The purpose of this work was to identify an unknown component which has been detected during the analysis of cyclic fatty acid monomers (CFAMs) in low erucic acid rapeseed oils (LEAR). A sample of crude LEAR was transformed into fatty acid methyl esters (FAMEs) and hydrogenated using PtO(2). The hydrogenated sample was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and the fraction containing the CFAMs transformed into picolinyl esters. Analysing these picolinyl derivatives by gas-liquid chromatography coupled to mass spectrometry (GC-MS) showed that the unknown product observed in LEAR is the 11,12-methylene-octadecanoic acid. This cyclic fatty acid was also found in crude LEAR and in the corresponding seeds but was not detected in crude soya and sunflower oils. As this acid is present in the same fraction as CFAMs, known to be formed during heat treatment, great care must therefore be taken for not including it when quantifying CFAMs. It is thus necessary to verify by mass spectrometry the structures of the CFAMs in the isolated cyclic fatty acid fraction prior to quantification.

224 Modification des teneurs en plasmalogènes et en acide docosahexaénoïque (DHA) dans les érythrocytes des patients glaucomateux

Introduction Les tissus nerveux comme le cerveau et la retine sont connus pour leur richesse en plasmalogenes (phospholipides ayant une liaison vinyl-ether) et en acides gras polyinsatures a longues chaines de la famille omega-3 (DHA). Des teneurs anormales en plasmalogenes et/ou en DHA ont ete decrites dans les membranes erythrocytaires de patients souffrant de desordres neurologiques et en particulier dans la maladie d’Alzheimer. Puisque les donnees de la litterature montrent un certain nombre de similarites physiopathologiques entre la maladie d’Alzheimer et les glaucomes, le but de ce travail a ete d’evaluer les teneurs en plasmalogenes et en DHA dans les membranes erythrocytaires de patients atteints de glaucomes. Materiels et Methodes Des echantillons de sang veineux ont ete collectes chez 41 sujets sains et 43 patients atteints de glaucome a angle ouvert. Le stade du glaucoma a ete determine suivant la classification de Hoddap et Parrish. Les erythrocytes ont ete isoles a partir du sang total puis laves avec du chlorure de sodium a 0,9 %. Apres extraction des lipides des membranes erythrocytaires, les phospholipides ont ete isoles par chromatographie sur cartouches de silice. Les teneurs en plasmalogenes et en DHA des phospholipides des membranes erythrocytaires ont ete determinees par chromatographie en phase gazeuse. Resultats Les teneurs en plasmalogenes ont ete significativement diminuees chez les patients glaucomateux, en particuliers ceux lies au C18 : 0, C18 : 1 n-9 et C18 : 1 n-7 en position sn -1 du glycerol. Les teneurs en DHA ont ete augmentees chez les patients atteints de glaucome debutant par rapport aux temoins alors qu’elles ont ete diminuees chez les patients atteints de glaucome modere et severe. Chez les patients glaucomateux, les indices du champ visuel ont ete correles de facon lineaire aux teneurs en DHA (r 2 respectifs de 0,87 et 0,99 pour le MD et le PSD). Discussion Les mecanismes responsables de la baisse des teneurs en plasmalogenes et en DHA dans les erythrocytes des patients glaucomateux restent indetermines. Conclusion D’autres travaux s’averent necessaires afin de determiner si ces evenements representent une des causes ou une consequence de la pathologie.

265 Modifications de la composition en acides gras du meibum et de la surface oculaire de patients souffrant de dysfonctionnement meibomien après traitement par minocycline

Introduction La structure du film lacrymal est fortement dependante des caracteristiques chimiques et physiques des lipides qui le composent. Nous avons precedemment observe une proportion plus forte en acides gras branches dans le meibum de patients souffrant de dysfonctionnement meibomien. Ce travail prospectif a eu pour objectif d’evaluer l’effet d’un traitement oral par minocycline associe a des soins de paupiere par comparaison a des soins de paupieres seuls sur les anomalies de la surface oculaire et sur la composition en acides gras du meibum de patients souffrant de dysfonctionnement meibomien. Materiels et Methodes Vingt patients souffrant de dysfonctionnement meibomien chronique ont ete suivis avant et apres 8 semaines de traitement oral par minocycline associe a des soins de paumieres (n = 10) ou de soins de paupieres seuls (n = 10). Nous avons evalue le break-up time (BUT), le degre de keratite, l’aspect physique du meibum excrete et preleve un echantillon de meibum a l’aide d’une bandelette de papier utilisee lors des tests de Schirmer chez ces patients.. Les acides gras du meibum ont ete directement derivatises par un agent trans-methylant puis analyses par chromatographie en phase gazeuse et par spectrometrie de masse. Resultats Le traitement par minocycline ameliore le BUT chez les patients souffrant de dysfonctionnement meibomien (p Discussion Cette etude confirme l’interet porte aux tetracyclines de synthese qui stabilisent le film lacrymal des patients presentant un dysfonctionnement meibomien. Les tetracyclines mais aussi les soins de paupiere ont montre qu’ils avaient un effet biologique en corrigeant partiellement des anomalies de la composition lipidique du meibum chez ces patients. Conclusion Les cyclines et la minocycline en particulier, s’averent complementaires des soins de paupieres en apportant un effet therapeutique supplementaire chez les patients souffrant de dysfonctionnement meibomien. L’isoC20 pourrait representer un marqueur du dysfonctionnement meibomien et de l’efficacite d’un traitement.

002 Évaluation de la toxicité des acides gras branchés sur des cellules conjonctivales en culture

Introduction Des travaux preliminaires ont montre une augmentation de la proportion en acides gras branches (acides gras dont la chaine est branchee par un ou plusieurs groupes methyl) dans le film lipidique lacrymal de patients souffrant de blepharite chronique. Or les acides gras branches ont prouve leur toxicite dans certains modeles cellulaires. Le but de notre travail a ete d’etudier les effets de certains acides gras branches sur des cellules conjonctivales en culture. Materiels et Methodes Des cellules conjonctivales humaines immortalisees ont ete incubees avec des solutions contenant les acides gras branches isoC16, isoC20, l’acide phytanique ou un acide gras lineaire : le C16 a des concentrations variant de 50 a 200 μm pendant 4 h ou 24 h. La toxicite des molecules a ete evaluee par la methode MTT (methylthiazol tetrazolium bromide), la croissance cellulaire, la production d’especes reactives de l’oxygene (dichlorodihydrofluoresceine), la permeabilite membranaire (iodure de propidium), la depolarisation mitochondriale (Mititracker) et l’apoptose (Hoechst). L’incorporation des acides gras dans les cellules a elle aussi ete etudiee. Resultats La respiration mitochondiale est diminuee significativement par l’IsoC20 et le C16 apres 4 h d’incubation et par l’isoC20 et l’acide phytanique apres 24h d’incubation (p Discussion L’isoC20, un des acides gras branches presents dans les lipides des larmes a raison de 3,5 % des acides gras totaux affecte la respiration mitochondriale. Cet effet ne semble pas lie au stress oxydatif, a la croissance cellulaire ou a la mort cellulaire. Il pourrait etre du a l’incorporation de cet acide gras au sein des membranes mitochondriales. Conclusion L’isoC16 et l’isoC20 n’ont pas le meme effet sur les cellules de la conjonctive. Cette difference pourrait etre liee a une incorporation preferentielle de l’isoC20 dans les membranes mitochondriales par rapport a l’iso C16.

Regio- and stereo-selectivity in the induction of peroxisome proliferation by substituted hexanoic acids

Summary— Quantitative structure‐activity relationship is an effective tool in order to predict drug potency. A similar approach is actually developed for peroxisome proliferation induced by substituted carboxylic acids issued from plasticizer metabolism in rats. The study is focused on acids found in rat urine after adipic diester dosings. Size, location of the substituted group and length of the chain have been studied. 3‐D structure has also been taken in account for 2‐ethyl hexanoic acids. The results obtained so far demonstrate that peroxisome proliferation potencies of the considered acids are modified according structure changes. At this time location of the group along the chain appears to be a predominant factor.